This invention relates to inhibition and detection of blood clot formation.
The resistance of thrombi to fibrinolysis induced by plasminogen activators is an impediment to the successful treatment of thrombotic diseases. Fibrinolytic resistance is evident in patients with acute thrombotic coronary occlusion, where treatment with plasminogen activators results in full coronary reperfusion in only 33-55% of patients at 90 minutes (Lincoff et al., 1995, Am. J. Cardiol. 75:871-876; Karagounis et al., 1992, J. Am. Coll. Cardiol. 19:1-10). The resistance of thrombi to lysis by plasminogen activators may be even more marked in patients with venous thromboembolism. In deep venous thrombosis treated with tissue plasminogen activator (TPA), nearly two thirds of patients have minimal or no significant lysis evident on repeat venography at 24 hours (Salzman et al., 1994, The epidemiology, pathogenesis and natural history of venous thrombosis, in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed., Philadelphia, Pa.: Lippincott, pp 1275-1296; Goldhaber et al., 1990, Am. J. Med. 88:235-240). In patients with pulmonary embolism, TPA restores blood flow within 24 hours to only about a third of occluded lung segments, as judged by serial perfusion scanning (The Urokinase Pulmonary Embolism Trial. A national cooperative study, 1973, Circulation 47:1-108; Goldhaber et al., 1986, Lancet 2:886-889). Improved thrombolysis may reduce mortality and morbidity associated with thrombotic disease.
The invention provides methods of improving therapeutic thrombolysis, detecting blood clots in vivo, and inhibiting clot formation using alpha-2 antiplasmin (xcex12AP) polypeptides.
To detect blood clot formation in a mammal, a diagnostically effective amount of a detectably labeled alpha-2 antiplasmin (xcex12AP) polypeptide is administered to the mammal, and association of the polypeptide with a blood clot determined. Association of the xcex12AP polypeptide with a vascular obstruction, e.g, via xcex12AP-fibrin crosslinking, is an indication of the presence blood clot formation at the site of the obstruction. Since xcex12AP crosslinks with fibrin in actively forming blood clots but not at the site of old (i.e., not actively forming) blood clots, the method is useful to characterize blood vessel obstructions as pre-existing or actively forming. Newly forming thrombi are detected by virtue of the formation of xcex12AP polypeptide-fibrin crosslinks mediated by activated factor XIIIa. Thus, the present method provides an advantage over conventional visual detection techniques such as scintiphotography and angiography with which such characterization is difficult or unachievable.
The polypeptides of the invention are substantially pure. Polypeptides or other compounds encompassed by the invention are said to be xe2x80x9csubstantially purexe2x80x9d when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The xcex12AP polypeptide preferably contains the amino acid sequence of X1QX2X3X4X5PLX6LLK (SEQ ID NO:1), wherein X1=N or A, X2=E or Q, X3=Q or K, X4=V or L, X5=P or S, and X6=T, S or A (the Q residue indicated in bold type is involved if xcex12AP-fibrin crosslinking). For example, the polypeptide comprises the amino acid sequence of xcex12AP13-24 NQEQVSPLTLLK (SEQ ID NO:2) or xcex12AP1-24 (MEPLGWQLTSGPNQEQVSPLTLLK; SEQ ID NO:16). Polypeptides derived from human xcex12AP-include those which contain a sequence that is 80-100% identical to the amino acid sequence of MEPLGXQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:3), where xe2x80x9cXxe2x80x9d represents a residue that can differ among human xcex12AP variants. Human N-terminal xcex12AP polypeptides include those which contain the amino acid sequence MEPLGWQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:4) or MEPLGRQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:5). Alternatively, the polypeptide may contain a sequence that is 80-100% identical to the amino acid MEPLDLQLMD GQAQQKLPPL SLLKLDNQEP GGQIAPKKAP EDCKLSPTPE QTRRLARAMM TFTTDLFSLV AQS (SEQ ID NO:6), corresponding to an N-terminal fragment of naturally-occurring bovine xcex12AP, or VDLPGQQPVS EQAQQKLPLP ALFKLDNQDF GDHATLKRSP GHCKSVPTAE ETRRLAQAMM AFTTDLFSLV AQT (SEQ ID NO:7), corresponding to an N-terminal fragment of naturally-occurring mouse xcex12AP . An xcex12AP polypeptide is a peptide with at least 80-100% sequence identity to a portion of a naturally-occurring xcex12AP protein but having a length that is shorter than the length of the naturally-occurring mature full-length xcex12AP protein. Human xcex12AP variants within the invention include those with the amino acid sequence of SEQ ID NO:11, 13, 15, or 17.
The invention also features methods of preventing the development of clots in patients at risk for thrombosis and methods of treating patients with thrombotic conditions such as stroke, myocardial infarction, pulmonary embolism, and deep venous thrombosis. For example, a method of inhibiting blood clot formation in a mammal is carried out by administering to the mammal a therapeutically effective amount of an xcex12AP polypeptide. A method of preventing and lysing blood clots is carried out by co-administering to a mammal a therapeutically effective amount of an xcex12AP polypeptide and a thrombolytic agent such as a plasminogen activator, e.g., tissue plasminogen activator (t-PA). Thrombolytic agents such as prourokinase, urokinase, streptokinase, staphylokinase, and vampire bat-derived plasminogen activator may be co-administered with an xcex12AP polypeptide to increase the effectiveness of the thrombolytic agent.
xcex12AP polypeptides and peptide mimetics thereof are useful in the diagnostic and therapeutic methods described above. Preferably the xcex12AP polypeptide is derived from the N-terminus of a mature, naturally-occurring mammalian xcex12AP protein. For example, the polypeptides may be derived from human, bovine, or mouse xcex12AP proteins, the amino acid sequences of which are shown in Tables 1-3, respectively (the first amino acid of the mature protein is indicated with an arrow).
The length of an xcex12AP polypeptide is preferably 12 to 250 amino acids, inclusive. More preferably, the length is 10 to 75 amino acids, inclusive, e.g., a polypeptide that is 12 amino acids in length and has an amino acid sequence of NQEQVSPLTLLK (SEQ ID NO:2).
The amino acid sequence of the polypeptide preferably contains the amino acid sequence of X1QX2X3X4X5PLX6LLK (SEQ ID NO:1), wherein X1=N or A, X2=E or Q, X3=Q or K, X4=V or L, X5=P or S, and X6=T, S or A. Examples of such polypeptides include those derived from human xcex12AP (e.g., SEQ ID NO:4 or 5) and those derived from bovine xcex12AP (e.g., SEQ ID NO:6). An xcex12AP polypeptide derived from mouse xcex12AP contains the amino acid sequence of SEQ ID NO:7. Shorter polypeptides such as those containing amino acids 1-24, 1-41, 10-24, and 13-24 (e.g., SEQ ID NO:2) of each of SEQ ID NO:4, 5, 6, or 7 are also useful to detect and treat thrombotic conditions.
Preferably, the xcex12AP polypeptide contains an amino acid sequence with 80-100% sequence identity to SEQ ID NO: 3, 4, 5, 6, or 7 in which a non-identical amino acid of the polypeptide is a conservative amino acid substitution and the polypeptide functions to inhibit xcex12AP-fibrin crosslinking. Sequence identity is measured using standard sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), with the default parameters as specified therein.
Peptide mimetics of an xcex12AP polypeptide are also within the invention. Preferably, the length of the peptide mimetic is 12 to 250 amino acids, inclusive, and contains the amino acid sequence of SEQ ID NO:1. For example, an xcex12AP peptide mimetic contains an amino acid sequence with 80-100% sequence identity to SEQ ID NO: 2; the non-identical amino acids of the polypeptide are conservative amino acid substitutions; and the polypeptide inhibits xcex12AP-fibrin crosslinking.
For diagnostic applications, the polypeptide is detectably labeled, e.g., the polypeptide is biotinylated or tagged with a radioisotope. In addition to administering xcex12AP polypeptides to a mammal to prevent clot formation or promote physiologic thrombolysis or improving the effectiveness of therapeutically-induced thrombolysis, the polypeptide can be used as targeting agents to deliver therapeutic agents to the site of a actively forming blood clot. For example, any of the xcex12AP polypeptides or peptide mimetic are linked to a therapeutic agent (e.g., a thrombolytic agent). Such chimeric compounds are recombinantly produced or cosynthesized. Thus, the invention includes a method of targeting a therapeutic agent to an actively developing thrombus in a mammal by administering to the mammal an N-terminal xcex12AP polypeptide linked to a therapeutic agent.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.